DNA Translocation and Unzipping through a Nanopore: Some Geometrical Effects

Abstract

This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

“…The first two peaks (denoted 〈 i 〉/〈 i 0 〉 3′ and 〈 i 〉/〈 i 0 〉 5′ ) are consistent with the dependence of the blockade depth on the orientation of the leading end of the DNA (3′ vs 5′) entering the pore. 4,52,53 The location of these two peaks agrees with previous measurements of dA 100 where two highly overlapping peaks were observed at these locations. 6 In contrast to earlier measurements we resolve the 3′ and 5′ events with a separation better than 3 σ .…”

Probing molecular pathways for DNA orientational trapping, unzipping and translocation in nanopores by using a tunable overhang sensor

“…These signatures and their molecular mechanisms have not been reported in the past, although the nucleic acid unzipping has been extensively studied 7 , 8 , 10 , 12 , 46 , 47 , 49 . Several previous studies were focused on the unzipping of hairpins 10 , 12 , 49 .…”

Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

“…The behaviors of ssDNA strands are rather different with respect to directionality when entering the protein channel, in terms of both event frequency and current blockage (38,40). Thus, the current signatures of both 5 0 -Btn-C 39 AP-18c6 14 (3′ entry) and 3 0 -Btn-C 39 AP-18c6 ω14 (5′ entry) were recorded with 5′-and 3 0 -Btn-C 40 as the references, respectively, in 1 M NaCl.…”

“…Also, there were many AP-18c6 events (30%) that could not enter the β barrel from the 5′ end (type 2b); yet, only ∼8% of the events had the same problem from the other terminus. These directionality effects can be explained by the tendency of ssDNA bases to tilt towards the 5′ end in a strongly confined environment, producing the "Christmas tree" geometry (40). As a result, DNA strands entering from the 5′ direction experience more friction from the protein, along with steric hindrance from the bulky 18c6-Na þ , causing more type 2b events to occur than during 3′ entry.…”

“…In the immobilization studies, Strep-Btn DNA (40 pmol, 200 nM) was added in the cell and more than 200 capture/release events were collected under 120 mV bias (trans versus cis) with a 10 kHz low pass filter, and 50 kHz data acquisition rate. Then, the same amount of Strep-Btn dC 40 control was added as an internal standard. For the translocation studies, ssDNA (2 nmol, 10 μM) was added and more than 1,000 events were collected for each voltage with a 100 kHz low pass filter, and 500 kHz data acquisition rate.…”